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Expression of markers for germ cells and oocytes in cow dermal fibroblast treated with 5-azacytidine and cultured in differentiation medium containing BMP2, BMP4 or follicular fluid

Published online by Cambridge University Press:  03 July 2017

José Jackson do Nascimento Costa ,
Glaucinete Borges de Souza ,
Joyla Maria Pires Bernardo ,
Regislane Pinto Ribeiro ,
José Renato de Souza Passos ,
Francisco Taiã Gomes Bezerra ,
Márcia Viviane Alves Saraiva  and
José Roberto Viana Silva
José Jackson do Nascimento Costa
Affiliation:
Biotechnology Nucleus of Sobral – NUBIS, Federal University of Ceara, CEP 62042–280, Sobral, CE, Brazil.
Glaucinete Borges de Souza
Affiliation:
Biotechnology Nucleus of Sobral – NUBIS, Federal University of Ceara, CEP 62042–280, Sobral, CE, Brazil.
Joyla Maria Pires Bernardo
Affiliation:
Biotechnology Nucleus of Sobral – NUBIS, Federal University of Ceara, CEP 62042–280, Sobral, CE, Brazil.
Regislane Pinto Ribeiro
Affiliation:
Biotechnology Nucleus of Sobral – NUBIS, Federal University of Ceara, CEP 62042–280, Sobral, CE, Brazil.
José Renato de Souza Passos
Affiliation:
Biotechnology Nucleus of Sobral – NUBIS, Federal University of Ceara, CEP 62042–280, Sobral, CE, Brazil.
Francisco Taiã Gomes Bezerra
Affiliation:
Biotechnology Nucleus of Sobral – NUBIS, Federal University of Ceara, CEP 62042–280, Sobral, CE, Brazil.
Márcia Viviane Alves Saraiva
Affiliation:
Biotechnology Nucleus of Sobral – NUBIS, Federal University of Ceara, CEP 62042–280, Sobral, CE, Brazil.
José Roberto Viana Silva*
Affiliation:
Biotechnology Nucleus of Sobral - NUBIS, Federal University of Ceara, Av. Comandante Maurocélio Rocha Ponte 100, CEP: 62.041–040, Sobral, CE, Brazil.
*
All correspondence to: J.R.V. Silva. Biotechnology Nucleus of Sobral – NUBIS, Federal University of Ceara, Av. Comandante Maurocélio Rocha Ponte 100, CEP: 62.041–040, Sobral, CE, Brazil. Tel:/Fax: +55 85 3611 8000. E-mail: [email protected]
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Summary

This study aims to investigate the effect 5-azacytidine (5-Aza) during induction of pluripotency in bovine fibroblasts and to evaluate the effects of BMP2, BMP4 or follicular fluid in the differentiation of reprogrammed fibroblasts in primordial germ cells and oocytes. It also analysis the mRNA levels for OCT4, NANOG, REX, SOX2, VASA, DAZL, cKIT, SCP3, ZPA and GDF9 after culturing 5-Aza treated fibroblasts in the different tested medium. Dermal fibroblasts were cultured and exposed to 0.5, 1.0 or 2.0 μM of 5-Aza for 18 h, 36 h or 72 h. Then, the cells were cultured in DMEM/F12 supplemented with 10 ng/ml BMP2, 10 ng/ml BMP4 or 5% follicular fluid. After culture, morphological characteristics, viability and gene expression were evaluated by qPCR. Treatment of skin fibroblasts with 2.0 μM 5-Aza for 72 h significantly increased expression of mRNAs for SOX2, OCT4, NANOG and REX. The culture in medium supplemented with BMP2, BMP4 or follicular fluid for 7 or 14 days induced formation of oocyte-like cells, as well as the expression of markers for germ cells and oocyte. In conclusion, treatment of bovine skin-derived fibroblasts with 2.0 μM 5-Aza for 72 h induces the expression of pluripotency factors. Culturing these cells in differentiation medium supplemented with BMP2, BMP4 or follicular fluid induces morphological changes and promotes expression of markers for germ cells, meiosis and oocyte.

Keywords

5-AzacytidineBMPFibroblastsFollicular fluidGene expression
Type
Research Article
Information
Zygote , Volume 25 , Issue 3 , June 2017 , pp. 341 - 357
Copyright
Copyright © Cambridge University Press 2017 

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